The isolation of preparative amounts of biologically active nucleic acid molecules has been a vexing problem in molecular biology. This is especially the case with regard to isolation of DNA for use in recombinant methodologies where it is required to be in sufficiently pure form to be digestible by restriction endonucleases, to be a good substrate for polymerases and topoisomerases, and to be suitable for use as a transfection or transformation agent.
Over the years, many methods have been developed to isolate nucleic acid molecules. However, those methods are typically tedious, require a high level of skill to perform, take extended periods of time to accomplish, require the processing of relatively large volumes of materials and often give variable results. In addition, most of the isolation techniques reported are costly in terms of equipment and materials. See, Gamper et al., DNA, 4:157-164 (1985); Yang et al., Meth. Enzol., 68:176-182 (1979); and Vogelstein et al., Proc. Natl. Acad. Sci. USA, 7:615-619 (1979).
To date, the art has applied several different technologies to the problem of preparing nucleic acids in quantities and purities sufficient for use in recombinant methodologies. Classically, the final step in the isolation of plasmid DNA is a cesium chloride-ethidium dye isopycnic gradient ultracentrifugation through a neutral or alkaline density gradient, gel electrophoresis, high-pressure liquid chromatography through RPC-5, alkaline extraction and column chromatography on methylated albumin kieselguhr, hydroxyapatite, or benzoylated naphthoylated DEAE-cellulose. See Gamper et al., DNA, 4:157-164 (1985); Yang et al., Meth. Enzol. 68:176-182 1979); Birnboim, Meth. Enzol., 100: 243-255 (1983); Mizutani, J. Chrom., 262:441-445 (1983); and the references cited therein.
Of particular interest to the present invention are methods wherein the nucleic acid to be isolated is adsorbed onto an insoluble silica matrix, e.g., particulate glass. While there are several reports of using the binding of nucleic acids to particulate glass as an isolation means, the physio-chemical mechanism(s) responsible for the binding phenomenon and the conditions under which it occurs are poorly characterized. Advances in the art have therefore proceeded on an empirical basis.
The use of adsorption onto glass as a means for isolating nucleic acids is based on the observation that both DNA and RNA bind to glass in highly concentrated aqueous salt solutions, i.e., salt concentrations of at least about 3 molar, and can be eluted therefrom by lowering the salt (ionic) concentration. While the pH value of the salt solution appears to have some effect on the adsorption process, that effect has not been characterized.
There have been several reports on the use of the glass-adsorption technique to isolate DNA from agarose gels. In each case, the salt solutions used to mediate the binding of the nucleic acids to the glass contained the buffering agent tris (hydroxymethyl) aminomethane at a concentration of less than 50 millimolar. Those solutions therefore had a low buffering capacity. See, Mizutani, J. Col. Inter. Sci., 93:270-273 (1983); Mizutani, J. Chrom., 262:441-445 (1983); Marko et al., Anal. Biochem., 121:382-387 (1982); Chen et al., Anal. Biochem., 101:339-341 (1980); Vogelstein et al., Proc. Natl. Acad. Sci. USA, 76:615-619 (1979); and Yang et al., Meth. Enzol., 68:176-182 (1979).
None of the previously reported methods of isolating nucleic acids by glass-adsorption has gained widespread acceptance by those skilled in the art of recombinant DNA technology. This is probably due to the inability of those methods to consistently separate DNA from sample contaminants such as RNA, protein and agarose. For instance, Marko et al., supra, reported that the buffered salt solution used to mediate DNA binding to glass was required to contain the chelating agent cyclohexanediamine tetraacetate (CDTA) in order to prevent binding of tRNA to glass and co-purification of the tRNA with the plasmid DNA.
From the foregoing it can be seen that there has been a long felt need by those practicing recombinant DNA technology for a reliable, rapid method for isolating nucleic acid molecules.